Use of untranslated region of osmotin gene to enhance transgene expression in plants

ABSTRACT

The present invention provides methods, vectors and gene constructs for enhancing expression of a recombinant nucleic acid sequence in transgenic plants and plant tissues. According to the present invention, nucleic acid sequences are obtained and/or derived from the 5′ and 3′ untranslated regions of genes encoding osmotin proteins and engineered to flank respective portions of a selected coding region of a vector. The vector construct may be introduced into plants and/or plant tissues through conventional procedures, resulting in enhanced expression of the selected coding region. In a preferred embodiment, the selected coding region is a chimeric gene or gene fragment expressing one or more proteins known to impart a level of insecticidal activity to a transgenic plant and/or plant tissue.

This application claims the benefit of U.S. Provisional Application No.60/416,142 filed on Oct. 4, 2002 and is being filed as a DivisionalApplication of U.S. Ser. No. 10/703,280 filed on Nov. 7, 2003 nowabandoned. The present invention relates to plant molecular biology andthe application of genetic engineering techniques to plants. Moreparticularly, the present invention provides DNA sequences, constructsand methods that are useful for enhancing the expression of recombinantgenes in plants.

FIELD OF THE INVENTION Background of the Invention

Recombinant DNA technology and genetic engineering have made itroutinely possible to introduce desired DNA sequences into plant cellsto allow for the expression of proteins of interest. For commerciallyviable transformation events, however, obtaining desired levels ofstable and predictable expression in important crops remainschallenging.

One method of expressing heterologous genes at desired levels in cropsinvolves manipulation of the regulatory mechanisms governing expressionin plants. The regulation may be transcriptional or post-transcriptionaland can include, for example, mechanisms to enhance, limit, or preventtranscription of the DNA, as well as mechanisms that limit or increasethe life span of an mRNA after it is produced. The DNA sequencesinvolved in these regulatory processes can be located upstream,downstream or even internally to the structural DNA sequences encodingthe protein product of a gene.

To regulate transcription in a transgenic plant, various types ofpromoters may be employed. Promoters can be used to control theexpression of foreign genes in transgenic plants in a manner similar tothe expression pattern of the gene from which the promoter wasoriginally derived. Generally, promoters are classified in twocategories. “Constitutive” promoters express in most tissues most of thetime. Expression from a constitutive promoter is more or less at asteady state level throughout development. Genes encoding proteins withhouse-keeping functions are often driven by constitutive promoters.Examples of constitutively expressed genes in maize include actin andubiquitin. (Wilmink et al., Plant Mol. Biol., 28:949-955, 1995).“Regulated” promoters are typically expressed in only certain tissuetypes (tissue specific promoters) or at certain times during development(temporal promoters).

Further improvements in transcription have been obtained in transgenicplants by placing “enhancer” sequences upstream (5′) of the promoter.Enhancer elements are cis-acting and increase the level of transcriptionof an adjacent gene from its promoter in a fashion that is relativelyindependent of the upstream position and orientation of the enhancer.Such sequences have been isolated from a variety of sources, includingviruses, bacteria and plant genes. One example of a well characterizedenhancer sequence is the octopine synthase (ocs) enhancer from theAgrobacterium tumefaciens, as described in U.S. Pat. Nos. 5,837,849,5,710,267 and 5,573,932, assigned to the assignee of the presentinvention. This short (40 bp) sequence has been shown to increase geneexpression in both dicots and monocots, including maize, by significantlevels. Tandem repeats of this enhancer have been shown to increaseexpression of the GUS gene eight-fold in maize. It remains unclear howthese enhancer sequences function. Presumably enhancers bind activatorproteins and thereby facilitate the binding of RNA polymerase II to theTATA box. WO95/14098 describes testing of various multiple combinationsof the ocs enhancer and the mas (mannopine synthase) enhancer whichresulted in several hundred fold increase in gene expression of the GUSgene in transgenic tobacco callus.

The use of a specific promoter, with or without one or more enhancers,however, does not necessarily guarantee desired levels of geneexpression in plants. In addition to desired transcription levels, otherfactors such as improper splicing, polyadenylation and nuclear exportcan affect accumulation of both mRNA and the protein of interest.Therefore, methods of increasing RNA stability and translationalefficiency through mechanisms of post-transcriptional regulation areneeded in the art.

With regard to post-transcriptional regulation, it is has beendemonstrated that certain 5′ and 3′ untranslated regions (UTRs) ofeukaryotic mRNAs play a major role in translational efficiency and RNAstability, respectively. For example, the 5′ and 3′ UTRs of tobaccomosaic virus (TMV) and alfalfa mosiac virus (AMV) coat protein mRNAs canenhance gene expression 5.4-fold and 3.0 fold in tobacco plants,respectively. (Zeyenko, FEBS Lett., November 14; 354(3):271-3 (1994)).The 5′ and 3′ UTRs of the maize alcohol dehydrogenase-1 (adh1) gene arerequired for efficient translation in hypoxic protoplasts.(Bailey-Serres et al., Plant Physiol., October; 112(2):685-95 (1996)).

Experiments with various 5′ UTR leader sequences demonstrate thatvarious structural features of a 5′UTR can be correlated with levelstranslational efficiency. Certain 5′ UTRs have been found to contain AUGcodons which may interact with 40S ribosomal subunits when it scans forthe AUG codon at the initiation site, thus decreasing the rate oftranslation. (Kozak, Mol. Cell. Biol. 7:3438 (1987); Kozak, J. CellBiol. 108, 209 (1989)). Further, the 5′UTR nucleotide sequences flankingthe AUG initiation site on the mRNA may have an impact on translationalefficiency. If the context of the flanking 5′UTR are not favorable, partof the 40S ribosomal subunits might fail to recognize the translationstart site such that the rate of polypeptide synthesis will be slowed.(Kozak, J. Biol. Chem. 266, 19867-19870 (1991); Pain, Eur. J. Biochem.236, 747-771 (1996)). Secondary structures of 5′UTRs (e.g., hairpinformation) may also hinder the movement of 40S ribosomal subunits duringtheir scanning process and therefore negatively impact the efficiency oftranslation. (Sonenberg et al., Nature 334:320 (1988); Kozak, Cell44:283-292, (1986)). The relative GC content of a 5′ UTR sequence hasbeen shown to be an indicator of the stability of the potentialsecondary structure, with higher levels of GC indicating instability.(Kozak, J. Biol. Chem. 266, 19867-19870 (1991). Longer 5′ UTRs mayexhibit higher numbers of inhibitory secondary structures. (Sonenberg etal., 1996). Thus, the translational efficiency of any given 5′ UTR ishighly dependent upon its particular structure, and optimization of theleader sequence has been shown to increase gene expression as a directresult of improved translation initiation efficiency. Furthermore,significant increases in gene expression have been produced by additionof leader sequences from plant viruses or heat shock genes. (Raju etal., Plant Science 94: 139-149 (1993)).

In addition to 5′ UTR sequences, 3′ UTR (trailer) sequences of mRNAs arealso involved in gene expression. 3′ UTRs (also known as polyadenylationelements or adenylation control elements) are known to control thenuclear export, polyadenylation status, subcellular targeting and ratesof translation and degradation of mRNA from RNases. In particular, 3′UTRs may contain one or more inverted repeats that can fold intostem-loop structures which act as a barrier to exoribonucleases, as wellas interact with proteins known to promoter RNA stability (e.g., RNAbinding proteins). (Barkan et al., A Look Beyond Transcription:Mechanisms Determining mRNA Stability and Translation in Plants,American Society of Plant Physiologists, Rockville, Md., pp. 162-213(1998)). Certain elements found within 3′ UTRs may be RNA destabilizing,however. One such example occurring in plants is the DST element, whichcan be found in small auxin up RNAs (SAURs). (Gil et al., EMBO J. 15,1678-1686 (1996)). A further destabilizing feature of some 3′ UTRs isthe presence of AUUUA pentamers. (Ohme-Takagi et al., Pro. Nat. Acad.Sci. USA 90 11811-11815 (1993)).

3′ UTRs have been demonstrated to play a significant role in geneexpression of several maize genes. Specifically, a 200 base pair 3′sequence has been shown to be responsible for suppression of lightinduction of the maize small m3 subunit of the ribulose-1,5-biphosphatecarboxylase gene (rbc/m3) in mesophyll cells. (Viret et al., Proc NatlAcad Sci USA. 91 (18):8577-81 (1994)). In plants, especially maize, thissequence is not very well conserved. One 3′ UTR frequently used ingenetic engineering of plants is derived from a nopaline synthase gene(3′ nos) (Wyatt et al., Plant Mol Biol 22(5):731-49 (1993)).

In certain plant viruses, such as alfalfa mosaic virus (AMV) and tobaccomosaic virus (TMV), the highly structured 3′ UTRs are essential forreplication and can be folded into either a linear array of stem-loopstructures which contain several high-affinity coat protein bindingsites, or a tRNA-like site recognized by RNA-dependent RNA polymerases.(Olsthoorn et al., EMBO J 1; 18(17):4856-64 (1999); Zeyenko et al.,1994)).

As of the date of the present invention, however, the use of 5′ and 3′UTRs to regulate the expression of recombinant nucleic acids intransgenic plants has not been wide-spread, mainly becauseoptimal/optimized UTR sequences have yet to be identified orcharacterized. Novel methods and compositions of matter for regulatinggene expression using optimal/optimized 5′ and 3′ UTRs are thereforeneeded in the art.

SUMMARY OF THE INVENTION

The present invention provides methods, vectors and gene constructs forenhancing expression of a recombinant nucleic acid sequence intransgenic plants and plant tissues. According to the present invention,nucleic acid sequences are obtained and/or derived from the 5′ and 3′untranslated regions of genes encoding osmotin proteins and engineeredto flank respective portions of a selected coding region of a vector.The vector construct may be introduced into plants and/or plant tissuesthrough conventional procedures, resulting in enhanced expression of theselected coding region. In a preferred embodiment, the selected codingregion is a chimeric gene or gene fragment expressing one or moreproteins known to impart a level of insecticidal activity to atransgenic plant and/or plant tissue.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram of the putative processing pathway of Toxin Aprotein and its proposed structure model. The molecular weight forunprocessed A0 protein and cleaved A1 and A2 polypeptides are indicated.(

): 87 amino acids at N-terminal end; (▪): 88 amino acids at C-terminalend of A1 polypeptide.

FIG. 2 provides diagrams of the Toxin A gene constructs used for planttransformation. Six different Toxin A gene fragments were insertedbetween a Cassaya Vein Mosaic Virus (CsVMV) promoter and Ti 15955plasmid ORF25 3′ sequences, respectively. RB: T-DNA right border; LB:T-DNA left border; Kan^(R): Kanamycin resistance gene. (

): 87 amino acids at N-terminal end; (▪): 88 amino acids at C-terminalend of A1 polypeptide. The designations for each Toxin A gene fragmentare shown in diagrams.

FIG. 3 is a Northern blot analysis showing the RNA expression patternsof transgenic Arabidopsis plants carrying six different Toxin Aconstructs. Above each lane are the names of the Toxin A gene fragmentscarried by the transgenic plants. The number of the plants with expectedRNA expression pattern (as shown in this blot) versus number of examinedplants for each construct are indicated below as n/N. MW: molecularweight. The position where non-specific binding of the probe toribosomal RNA occurs is indicated by an arrow.

FIG. 4 is a Western blot analysis of Toxin A expression in transgenicArabidopsis plants. (A): Lane 1: Recombinant E. coli strain; Lane 2:Transgenic plant with CsVMV-GUS-ORF25 gene construct; Lane 3-10:Transgenic plants with construct pDAB7031. (B): Lane 1: Recombinant E.coli strain; Lane 2-6: Transgenic plants with construct pDAB7036. Thebands below the A2 protein are the antibody cross-reacted background ofArabidopsis plants.

FIG. 5 is a Western blot analysis of A1 protein expression in transgenicArabidopsis plants. (A): Lane 1: Recombinant E. coli strain; Lanes 2-6:Transgenic plants with construct pDAB7035; Lane 7: Transgenic plant withGUS gene construct. (B): Lane 1: Recombinant E. coli strain; Lanes 2-6:Transgenic plants with construct pDAB7033; Lane 7: Transgenic plant withGUS gene construct. (C): Lane 1: Recombinant E. coli strain; Lanes 2-6:Transgenic plants with construct pDAB7034; Lane 7: Transgenic plant withGUS gene construct. The bands below the A2 protein are the antibodycross-reacted background of Arabidopsis plants.

FIG. 6 shows sequences and structural features of 5′ and 3′ UTRs of thetobacco osmotin gene. (A): DNA sequence of osmotin 5′ UTR (SEQ. ID.NO. 1) and its A/T content; (B) Computer-predicted RNA secondarystructure of osmotin 3′ UTR sequences.

FIG. 7 provides diagrams of the modified Toxin A gene constructs usedfor plant transformation. The 5′ and 3′ UTR sequences of tobacco osmotingene (

) were added to the both ends of Toxin A genes in constructs pDAB7031,pDAB7033, and pDAB7032 (FIG. 2). The resultant constructs are designatedpDAB7026, pDAB7027, and pDAB7028 as indicated on the right. CsVMV:Cassaya Vein Mosaic Virus promoter; ORF 25: ORF25 3′ sequences of Ti15955 plasmid. RB: T-DNA right border; LB: T-DNA left border; Kan^(R):Kanamycin resistance gene. ▪): 87 amino acids at N-terminal end; (

): 88 amino acids at C-terminal end of A1 polypeptide. The designationsfor each Toxin A gene fragment are shown in diagrams. The names for eachconstruct are indicated on right.

FIG. 8 provides an analysis of Toxin A expression of transgenicArabidopsis plants: (A) Western blot analysis of A2 protein expressionin transgenic plants with construct pDAB7028. Lanes 1-4, and Lanes 6-8:transgenic plants; Lane 5: recombinant E. coli strain. (B) SDS-PAGE gelanalysis of Toxin A expression in transgenic plants with constructpDAB7026. Lane 1: high expresser of Toxin A gene; Lanes 2-4: three lowexpressers of Toxin A gene; Lane 5: Transgenic plants with GUS geneconstruct. The positions of A0 and A1 proteins are indicated by arrows.Ten ug of total leaf protein were loaded into each lane.

FIG. 9 provides multi-generation analyses of insect resistance intransgenic line 7026-057. The number of active plants in each givenprogeny family are indicated as n/N (active plants/total examinedplants) and as percentage of total examined plants (shown inparenthesis). All the active plants shown here were high expressers andhad 100% insect mortality. The number of generation for each progenyfamily is indicated on left.

FIG. 10 is a comparative analysis of Toxin A RNA expression between highand low expressers of T1 progeny of line 7026-190. (A) Northern analysisof Toxin A RNA expression. Lanes 1-4: four T₁ plants with low Toxin Aexpression level (<50 ppm). Lanes 5-8: four T₁ plants with high Toxin Aexpression level (>1,400 ppm). (B) Ethidium bromide staining of RNA gel.

FIG. 11 illustrates the activity of Toxin A-expressing transgenic plantsagainst THW with different ages. Sixteen larvae at each instar stagewere used to test the activity of Toxin A-expressing plants. Plantstransformed with GUS construct were used as negative control. The datashown above are the average of two replicate experiments.

FIG. 12 is a map of plasmid pDAB1542.

FIG. 13 is a map of plasmid pKA882.

FIG. 14 is a map of plasmid pDAB7013

FIG. 15 is a map of plasmid pDAB7020.

FIG. 16 is a map of plasmid pDAB6001.

FIG. 17 is a map of plasmid pDAB7002.

FIG. 18 is a map of plasmid pDAB7021.

FIG. 19 is a map of plasmid pDAB7026.

FIG. 20 is a map of plasmid pDAB7031.

FIG. 21 is the nucleotide sequence of the full length A0 osmotin gene(SEQ. ID. No. 5).

FIG. 22 is the nucleotide sequence of the A0 osmotin gene with anN-terminal truncation (SEQ. ID. No. 6).

FIG. 23 is the nucleotide sequence of the A0 osmotin gene withC-terminal truncation (SEQ. ID. No. 7).

FIG. 24 is the nucleotide sequence of the full length A1 osmotin gene(SEQ. ID. No. 8).

FIG. 25 is the nucleotide sequence of the A1 osmotin gene withN-terminal and C-terminal truncations (SEQ. ID. No. 9).

FIG. 26 is the nucleotide sequence of the A2 osmotin gene (SEQ. ID. No.10).

FIG. 27 is the protein sequence of Photorabdus Toxin A (SEQ. ID. No. 4).

FIG. 28 (Table 1) summarizes a quantitative analysis of proteinexpression of transgenic plants carrying various Toxin A constructs.

FIG. 29 (Table 2) provides a comparison of protein expression andinsecticidal activity between transgenic plants carrying Toxin A andosmotin-Toxin A constructs.

FIG. 30 (Table 3) summarizes bioassay results of 274 transgenicArabidopsis lines transformed with construct pDAB7026.

FIG. 31 (Table 4) provides data regarding average insect mortality ofhigh and low expressions of T₁ transgenic plants transformed withconstruct pDAB7026.

FIG. 32 (Table 5) provides an analysis of protein expression and insectresistance of T₁ progeny of transgenic lines transformed with constructpDAB7026 which showed high Toxin A expression, but low activity at theT₀ generation.

FIG. 33 (Table 6) provides a multi-generation analysis of insectresistance demonstrated by transgenic line 7026-001.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides compositions and methods for geneticallymodifying cells, tissues, or organisms using 5′ and/or 3′ UTR regionsisolated or derived from a tobacco osmotin gene. The 5′ and/or 3′ UTRregions of the present invention, when engineered to flank a structuralnucleic acid of interest, improve mRNA stability and/or increasetranslational efficiency of the structural gene of interest in atransgenic plant. Thus, the present invention will facilitate thegenetic engineering of plants to express phenotypes of economic orinvestigative value.

Osmotin is a small (24 kDa), basic, pathogenesis-related protein that ishighly accumulated during adaptation of tobacco (Nicotiana tabacum)cells to osmotic stress, where it accounts for about 12% total solubleprotein. In tobacco, two osmotins have been described, osmotin-I: awater-soluble form, and osmotin-II: a detergent-soluble, relativelyprotease resistant form. Both osmotins from tobacco have a molecularweight of about 24 kD, show a large amino-acid sequence identity, aswell as similarity with a 24 kD osmotin-like protein from tomato(Lycopersicon esculentum), and to other proteins, including thaumatinfrom Thaumatococcus daniellii, pathogenesis-related protein S (PR-S)from tobacco, and a bifunctional maize trypsin/α-amylase inhibitor.

The expression of the osmotin gene is induced by various stress-relatedsignals, such as those resulting from water deficit, salinity, viralinfection and wounding. (See Singh et al., Plant Physiol., 85, 529-536(1987); Singh et al., Plant Physiol., 90, 1096-1101 (1989); Singh etal., In NATO ASI Series, G19, pp. 67-87 “Environmental Stress inPlants”, J. H. Cherry, ed (1989); LaRosa et al., Plant Physiol., 91,855-861 (1989); Meeks-Wagner et al., Plant Cell, 1, 25-35 (1989);Grosset et al., Plant Physiol., 92, 520-527 (1990); Neale et al., PlantCell, 2, 673-684 (1990); Roberts et al., J. Gen. Microbiol., 136,1771-1778 (1990); Stintzi et al., Physiol. Mol. Plant Pathol., 38,137-146 (1991); Woloshuk et al., Plant Cell, 3, 619-628 (1991); Singh etal., Plant Physiol. 79, 126-137 (1985); Richardson et al., Nature, 327,432-434 (1987); Bol, In Temporal and Spatial Regulation of Plant Genes,D. P. S. Verma and R. B. Goldberg eds (New York: Springer-Verlag) pp.201-221 (1988); Brederode et al., Mol. Biol., 17, 1117-1125 (1991);Linthorst, Crit. Rev. Plant Sci., 10, 123-150 (1991); LaRosa et al.,Plant Physiol., 79, 138-142 (1985); La Rosa et al., Plant Physiol., 85,174-185 (1987); Singh et al., Proc. Natl. Acad. Sci. USA, 84, 739-743)(1987)).

Both osmotin mRNA and osmotin protein are extremely stable. With regardto the present invention, the structural features of the 5′ and 3′ UTRsof the tobacco osmotin gene are consistent with the highly stable natureof the osmotin mRNA and protein: 1) its 5′ UTR sequence is highlyAT-rich, which allows 40S ribosomal subunits to easily scan to its startcodon to initiate translation (See FIG. 6A); and 2) its 3′ UTR sequencescan form a strong stem-loop secondary structure that may effectivelyblock the degradation from RNase (FIG. 6B).

According to preferred embodiments of the present invention, one or bothof 5′ and 3′ UTR regions isolated or derived from an osmotin gene aregenetically engineered to flank a structural gene of interest encoding aprotein which is expressed recombinantly in a plant, plant cell or planttissue. Preferably, the osmotin gene is isolated or derived from atobacco osmotin gene.

The following definitions are provided in order to remove clarify theintent, scope and usage of certain terms in the Specification and Claimsherein.

The term “chimeric gene construct”, as used herein, means a recombinantnucleic acid comprising genes or portions thereof from more than oneorganism.

A “deletion”, as used herein, refers to a change in either amino acid ornucleotide sequence in which one or more amino acid or nucleotideresidues, respectively, are absent.

A 5′ and/or 3′ osmotin UTR of the present invention is said to be“functionally linked” to a structural nucleic acid sequence of interestif these elements are situated in relation to another such that the 5′and 3′ osmotin UTR influences mRNA stability, translational efficiencyof transcription products of the structural nucleic acid sequence ofinterest.

The term “heterologous gene”, as used herein, means a gene encoding aprotein, polypeptide, RNA, or a portion of any thereof, whose exactamino acid sequence is not normally found in the host cell, but isintroduced by standard gene transfer techniques.

The term “homology”, as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence is one that atleast partially inhibits an identical sequence from hybridizing to atarget nucleic acid; it is referred to using the functional term“substantially homologous.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or probe will compete for and inhibitthe binding (i.e., the hybridization) of a completely homologoussequence or probe to the target sequence under conditions of lowstringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target sequence which lacks even apartial degree of complementarity (e.g., less than about 30% identity);in the absence of non-specific binding, the probe will not hybridize tothe second non-complementary target sequence.

The terms “identity” and “similarity”, as used herein, and as known inthe art, are relationships between two polypeptide sequences or twopolynucleotide sequences, as determined by comparing the sequences. Inthe art, identity also means the degree of sequence relatedness betweentwo polypeptide or two polynucleotide sequences as determined by thematch between two strings of such sequences. Both identity andsimilarity can be readily calculated (Computational Molecular Biology,Lesk, A. M., ed., Oxford University Press. New York (1988);Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,Academic Press, New York (1993); Computer Analysis of Sequence Data,Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, NewJersey (1994); Sequence Analysis in Molecular Biology, von Heinje, G.,Academic Press (1987); and Sequence Analysis Primer, Gribskov, M. andDevereux, J., eds., M Stockton Press, New York (1991)). Methods commonlyemployed to determine identity or similarity between two sequencesinclude, but are not limited to those disclosed in Carillo, H., andLipman, D., SIAM J. Applied Math., 48 : 1073 (1988). Preferred methodsto determine identity are designed to give the largest match between thetwo sequences tested. Methods to determine identity and similarity arecodified in computer programs. Typical computer program methods todetermine identity and similarity between two sequences include: GCGprogram package (Devereux, J., et al., Nucleic Acids Research 12 (1):387 (1984)), BLASTP, BLASTN, FASTA and TFASTA (Atschul, S. F. et al., J.Mol. Biol. 215: 403 (1990)).

An “insertion” or “addition”, as used herein, refers to a change in anamino acid or nucleotide sequence resulting in the addition of one ormore amino acid or nucleotide residues, respectively, as compared to thenaturally occurring molecule.

The term “modified expression”, as used herein, means expression in atransgenic plant which is genetically engineered to have one or both ofthe 5′ and 3′ osmotin UTRs of the present invention flanking therespective regions of a heterologous structural gene of interest whereinthe mRNA levels, protein levels or enzyme specific activity of thestructural gene of interest have been altered relative to 1) a nativeversion of the plant, or 2) a transgenic plant harboring the structuralgene of interest but not including the one or both of the 5′ and 3′osmotin UTRs as flanking regions thereof.

By “non-native phenotype”, as used herein, it is meant a traitoccurring, or influenced by, expression of recombinant DNA in a plant.

As used herein, the term “recombinant nucleic acid” refers to nucleicacid that has been derived or isolated from any source, that may besubsequently chemically altered, and later introduced into a transgenicplant. An example of recombinant nucleic acid “derived” from a source,would be a DNA or RNA sequence that is identified as a useful fragmentwithin a given organism, and which is then chemically synthesized inessentially pure form. An example of such DNA “isolated” from a sourcewould be a useful DNA sequence that is excised or removed from saidsource by chemical means, e.g., by the use of restriction endonucleases,so that it can be further manipulated, e.g., amplified, for use in theinvention, by the methodology of genetic engineering.

The term “stringency” is used herein to describe the conditions oftemperature, ionic strength, and the presence of other compounds such asorganic solvents, under which nucleic acid hybridizations are conducted.Those skilled in the art will recognize that “stringency” conditions maybe altered by varying the parameters just described either individuallyor in concert. With “high stringency” conditions, nucleic acid basepairing will occur only between nucleic acid fragments that have a highfrequency of complementary base sequences (for example, hybridizationunder “high stringency” conditions may occur between homologs with about85-100% identity, preferably about 70-100% identity). With mediumstringency conditions, nucleic acid base pairing will occur betweennucleic acids with an intermediate frequency of complementary basesequences (for example, hybridization under “medium stringency”conditions may occur between homologs with about 50-70% identity). Thus,conditions of “weak” or “low” stringency are often required with nucleicacids that are derived from organisms that are genetically diverse, asthe frequency of complementary sequences is usually less.

The term “structural nucleic acid sequence of interest”, as used herein,means a sequence of DNA, RNA or synthetic nucleotides that code for aprotein. The term “structural nucleic acid of interest” is usedinterchangeably herein with the term “structural gene of interest”.

As used in the present application, the term “substantial sequencehomology” is used to indicate that a nucleotide sequence (in the case ofDNA or RNA) or an amino acid sequence (in the case of a protein orpolypeptide) exhibits substantial, functional or structural equivalencewith another nucleotide or amino acid sequence. Any functional orstructural differences between sequences having substantial sequencehomology will be de minimis; that is they will not affect the ability ofthe sequence to function as indicated in the present application.Sequences that have substantial sequence homology with the sequencesdisclosed herein are usually variants of the disclosed sequence, such asmutations, but may also be synthetic sequences.

A “substitution”, as used herein, refers to the replacement of one ormore amino acids or nucleotides by different amino acids or nucleotides,respectively.

“Transformation”, as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the host cell being transformedand may include, but is not limited to, viral infection,electroporation, lipofection, and particle bombardment. Such“transformed” cells include stably transformed cells in

which the inserted DNA is capable of replication either as anautonomously replicating plasmid or as part of the host chromosome. Theyalso include cells which transiently express the inserted DNA or RNA forlimited periods of time.

“Nucleic acid sequence”, as used herein, refers to a polymer ofnucleotides in which the 3′ position of one nucleotide sugar is linkedto the 5′ position of the next by a phosphodiester bridge. In a linearnucleic acid strand, one end typically has a free 5′ phosphate group,the other a free 3′ hydroxyl group. Nucleic acid sequences may be usedherein to refer to oligonucleotides, or polynucleotides, and fragmentsor portions thereof, and to DNA or RNA of genomic or synthetic originthat may be single- or double-stranded, and represent the sense orantisense strand.

A promoter nucleic acid sequence is said to be “operably linked” to astructural nucleic acid sequence of interest if the two are situatedsuch that the promoter nucleic acid sequence influences thetranscription of the structural nucleic acid sequence of interest. Forexample, if the structural nucleic acid sequence codes for theproduction of a protein, the promoter nucleic acid sequence would beoperably linked to the structural nucleic acid sequence if the promoternucleic acid sequence affects the expression of the protein product fromthe structural nucleic acid sequence.

“Transgenic plant”, as used herein, refers to a plant that contains aforeign nucleotide sequence inserted into either its nuclear genome ororganellar genome.

The term “derivative”, as used herein, refers to a modification of thenative nucleic acid sequence of a 5′ and/or 3′ tobacco osmotin UTR.Illustrative of such modifications with regard to a 3′ tobacco osmotinUTR, would be the substitution, insertion, and/or deletion of one ormore bases relating to a nucleic acid sequence of a 3′ tobacco osmotinUTR that preserve, slightly alter, or increase the protective functionof one or more stem loop structures of the 3′ UTR against RNasedegradation. Such derivatives can be readily determined by one skilledin the art, for example, using sequence information to determineinverted repeats and using computer modeling techniques for predictingand optimizing optimal and suboptimal secondary structures, examples ofwhich are discussed herein. A derivative of a 5′ tobacco osmotin UTRmay, for example, comprise a substitution, insertion, and/or deletion ofone or more bases relating to a nucleic acid sequence of a 5′ tobaccoosmotin UTR that a) increase the AT (or AU) content; b) provide anoptimized nucleotide context surround the AUG codon of the 5′ end of thegene of interest; and/or c) do not add secondary structures whichinhibit the scanning process of 40S ribosomal subunits. The term“derivative” thus also includes nucleic acid sequences havingsubstantial sequence homology with the specifically disclosed regulatorysequences, such that they are able to have the disclosed effect onexpression.

Computer modeling techniques for use in predicting/evaluating 5′ and 3′UTR derivatives of the present invention include, but are not limitedto: MFold version 3.1 available from Genetics Corporation Group,Madison, Wis. (see Zucker et al., Algorithms and Thermodynamics for RNASecondary Structure Prediction: A Practical Guide. In RNA Biochemistryand Biotechnology, 11-43, J. Barciszewski & B. F. C. Clark, eds., NATOASI Series, Kluwer Academic Publishers, Dordrecht, N L, (1999); Zuckeret al., Expanded Sequence Dependence of Thermodynamic ParametersImproves Prediction of RNA Secondary Structure. J. Mol. Biol. 288,911-940 (1999); Zucker et al., RNA Secondary Structure Prediction. InCurrent Protocols in Nucleic Acid Chemistry S. Beaucage, D. E.Bergstrom, G. D. Glick, and R. A. Jones eds., John Wiley & Sons, NewYork, 11.2.1-11.2.10, (2000)), COVE (RNA structure analysis usingcovariance models (stochastic context free grammar methods)) v.2.4.2(Eddy & Durbin, Nucl. Acids Res. 1994, 22: 2079-2088) which is freelydistributed as source code and which can be downloaded from theinternet, and FOLDALIGN, also freely distributed and available fordownloading from the internet (see Finding the most significant commonsequence and structure motifs in a set of RNA sequences. J. Gorodkin, L.J. Heyer and G. D. Stormo. Nucleic Acids Research, Vol. 25, no. 18 pp3724-3732, 1997; Finding Common Sequence and Structure Motifs in a setof RNA Sequences. J. Gorodkin, L. J. Heyer, and G. D. Stormo. ISMB 5;120-123, 1997).

Native, optimized, fragmented or otherwise modified versions of the 5′tobacco osmotin UTR may be used to flank the 5′ region one or morestructural genes of interest in a construct. The native sequence of the5′ tobacco osmotin UTR is as follows:tatccaacaacccaacttgttaaaaaaaatgtccaacaac (SEQ. ID No. 1). (Nelson etal., Analysis of structure and transcriptional activity of an osmotingene. Plant Mol. Bio. 19:577-588 (1992)).

One skilled in the art will readily be able ascertain usable derivationsof the native sequence. In one preferred embodiment, exemplified herein,the single “atg” codon has been modified to “att” such that 40Sribosomal subunits will not be hindered by the semblance of aninitiation codon in the 5′ UTR. According to this embodiment, thenucleic acid sequence of the native 5′ tobacco osmotin UTR has beenmodified to

(SEQ. ID No. 2) tatccaacaacccaacttgttaaaaaaaatttccaacaac where thesingle base change is shown with underlining.

Native, optimized, fragmented or otherwise modified versions of the 3′tobacco osmotin UTR may also be used to flank the 3′ region of one ormore structural genes of interest in a construct. The published nativesequence of the 3′ tobacco osmotin UTR is:

(SEQ. ID No. 3) agtggctatttctgtaataagatccaccttttggtcaaattattctatcgacacgttagtaagacaatctatttgactcgtttttatagttacgtactttgtttgaagtgatcaagtcatgatctttgctgtaataaacctaagacctgaataagagtcacatatgtatttttgtcttgatgttatatagatcaataatgcatttggattatcgtttttatattgtttttcttttgaagttttagtaaa gtcttaagctt. (Nelson et al.(1992).

In most cases, sequences having 95% homology to the 5′ and 3′ tobaccoosmotin UTR sequences specifically disclosed herein will function asequivalents, and in many cases considerably less homology, for example75% or 80%, will be acceptable. Locating the parts of these sequencesthat are not critical may be time consuming, but is routine and wellwithin the skill in the art.

To modify the subject 5′ and 3′ UTR sequences in accordance with theteachings of this invention, exemplary techniques include those forpolynucleotide-mediated, site-directed mutagenesis as well as well knowntechniques for the use of restriction enzymes, PCR amplification andligase to modify and/or join existing nucleic acid molecules. (See,e.g., Zoller et al., DNA, 3:479-488 (1984); Higuchi et al., Nucl. AcidsRes., 16:7351-7367 (1988); Ho et al., Gene, 77:51-59 (1989); Horton etal., Gene, 77:61 (1989); PCR Technology: Principles and Applications forDNA Amplification, (ed.) Erlich (1989); and U.S. Pat. No. 6,271,360 toMetz et al., Single-stranded oligodeoxynucleotide mutational vectors(issued Aug. 7, 2001)). In a preferred embodiment of the invention, oneor more stem loop structures are added to SEQ. ID. No. 2 to providefurther protection against mRNA degradation. In one aspect of thisembodiment, the additional stem loop structures are derived through PCRamplification of all or part of SEQ. ID. No. 3. Stem loop structures mayalso be synthesized independently of SEQ. ID. No. 3. In a furtherembodiment of the invention, one or more existing stem loop structureswithin SEQ. ID. No. 3 are deleted, for example, by the use ofsite-specific restriction enzymes known to those skilled in the art.

Preferably, the 5′ and 3′ tobacco osmotin UTRs of the present inventionare used in conjunction with one another with regard to flanking theappropriate regions of one or more structural genes of interest. Thepresent invention, however, is not so limited. One or both of the 5′ or3′ tobacco osmotin UTRs of the present invention may thus be used, forexample, in conjunction with a UTR native to the structural gene(s) ofinterest, heterologous to the structural gene(s) of interest and thetobacco osmotin gene, or in addition to such a native or heterologousUTR.

The 5′ and 3′ osmotin UTRs for use in the present invention can beisolated from tobacco tissues or cells by means of nucleic acidhybridization techniques known in the art using, for example, thenucleotide sequences disclosed herein or portions thereof ashybridization probes. Such probes may consist of the entire osmotin geneor portions thereof, including the 5′ and 3′ UTRs identified herein. Thesubject osmotin 5′ and 3′ UTRs may also be synthetic and obtained usingthe above described sequences and nucleic acid synthesis techniquesknown in the art. Further, osmotin-encoding nucleotide sequences can beobtained from pOC cDNA clones as described by Singh et al., PlantPhysiol. 90:1096-1101 (1989).

Other plants from which osmotin genes can be isolated are inter alia,millet, soybean cotton, tomato and potato, described by Singh et al(1987), and King et al., Plant. Mol. Biol. 10, 401-412 (1988). It isfurther contemplated that UTRs from genes encoding osmotin-like proteinsfrom other plants than those mentioned above, such as maize, can be usedin accordance with the present invention as can reasonably be expectedto have similar homology to osmotin UTRs from tobacco.

The structural nucleic acid sequence of interest is operably linked to5′ and/or 3′ UTR regions isolated or derived from an osmotin gene byknown cloning techniques. The structural nucleic acid sequence ofinterest may be heterologous or homologous to the genes nativelypresently in the recipient plant, plant cell(s), or plant tissue. Ineither case, the 5′ and 3′ osmotin UTRs of the present invention areuseful for regulating the translational efficiency of a nucleic acidsequence of interest so as to: increase the half-life of transcribedmRNA; and/or express the protein encoded by the structural nucleic acidsequence of interest in greater abundance in plant tissue than would beexpressed without use of the 5′ and/or 3′ osmotin UTR(s) of the presentinvention. It is further specifically contemplated herein that thepresent invention is used in a gene construct engineered such that theprotein encoded by the structural nucleic acid sequence of interest isexpressed only in certain preferred tissue of a plant, such as theroots, leaves or stems, and not in the seed.

The present invention is generally applicable to the expression ofstructural genes of interest in both monocotyledonous and dicotyledonousplants. This invention is thus suitable for any member of themonocotyledonous (monocot) plant family including, but not limited to,maize, rice, barley, oats, wheat, sorghum, rye, sugarcane, pineapple,yams, onion, banana, coconut, and dates. A preferred application of thepresent invention is in the production of transgenic maize plants.Dicotyledonous (dicot) species for use with the present inventioninclude, but are not limited to, tobacco, tomato, sunflower, cotton,sugarbeet, potato, lettuce, melon, soybean and canola (rapeseed).

The structural nucleic acid sequence of interest used in constructs ofthe present invention may be any nucleic acid sequence that providesfor, or enhances, a beneficial feature of a resultant transgenic plant.Particularly useful nucleic acid sequences are those that encodeproteins or antisense RNA transcripts in order to promote increasednutritional values, higher yields, tolerance to herbicides, insects, ordiseases, and the like. More preferably, the nucleic acid sequences willbe useful genes which are inherently unstable due to their relativelylarge size (at least 4-5 kb in length), which is known to render thegenes more susceptible to physical, chemical, or enzymatic degradation.Genes inherently unstable due to their size include insecticidal genesfrom Xenorhabdus (see U.S. Pat. No. 6,048,838) and Photorabdus (e.g.,Toxin A as discussed herein).

In one preferred embodiment of the present invention, one or morestructural nucleic acids of interest are flanked by one or more osmotinUTRs of the present invention which have been “stacked” in relation toone another in a particular crop variety. By use of the terms “stacked”or “stacking”, it is meant herein that multiple structural genes ofinterest, each structural gene of interest preferably conferring acommercially desirable trait, have been transgenically introduced into asingle crop variety (inbred or hybrid). For example, a corn hybrid withstacked genes might contain genes for the insect resistance (e.g., Cry1FB.t. genes) as well as herbicide resistance genes (e.g., glyphosateresistance genes).

In another preferred embodiment, one or more of the osmotin UTRs of thepresent invention are functionally linked to a Toxin A gene fromPhotorabdus, which is then stacked with one or more insecticide and/orherbicide resistance genes in a single crop variety. Preferably, but notnecessarily, the insecticide gene(s) will be from a Bacillusthuringiensis or Xenorhabdus spp., and the herbicide gene(s) will be oneor more of a glufosinate, glyphosate, imidazolinone, or 2.4-D orsulfonyl urea resistance genes. Of course, any of the “stacked”insecticide or herbicide genes may be functionally linked to the osmotinUTRs of the present invention.

The structural nucleic acid sequence of interest may be derived in wholeor in part from a bacterial genome or episome, eukaryotic genomic,mitochondrial or plastid DNA, cDNA, viral nucleic acid, or chemicallysynthesized nucleic acid. It is contemplated that the structural nucleicacid sequence of interest may contain one or more modifications ineither the coding region which could affect the biological activity orthe chemical structure of the expression product, the rate ofexpression, or the manner of expression control. Such modificationsinclude, but are not limited to, mutations, insertions, deletions,rearrangements and substitutions of one or more nucleotides. Thestructural nucleic acid sequence of interest may constitute anuninterrupted coding sequence or it may include one or more introns,bounded by the appropriate plant-functional splice junctions. Thestructural nucleic acid sequence of interest may be a composite ofsegments derived from a plurality of sources, naturally occurring orsynthetic. The structural nucleic acid sequence of interest may alsoencode a fusion protein, so long as the experimental manipulationsmaintain functionality in the joining of the coding sequences.

In carrying out the present invention, cloning techniques are employedso as to obtain a vector containing the 5′ and/or 3′ osmotin UTRsflanking the structural gene of interest for subsequent introductioninto desired host cells. The 5′ and 3′ osmotin UTRs, structural nucleicacid sequence of interest, and any desired promoters, enhancers,selectable markers, etc. may thus be isolated and cloned into vectorsusing standard cloning procedures in the art, such as those described byJ. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press (2d ed., 1989), and Ausubel, F. M. et al. (1989)Current Protocols in Molecular Biology, John Wiley & Sons, New York,N.Y. both of which are hereby incorporated by reference.

A wide variety of cloning vectors are available, or can be prepared,where the cloning vector includes a gene construct functional in adesired plant species. Illustrative vectors include, for example,pBR322, pUC series, pACYC184, Bluescript series (Stratagene), and thelike. Such vectors are thus commercially available or can be readilyprepared for transformation of plant cells. In general, plasmid or viralvectors will contain nucleic acid sequences necessary for bothmaintenance and expression of a heterologous DNA sequence in a givenhost. Selection of appropriate elements to optimize expression in anyparticular species is a matter of ordinary skill in the art utilizingthe teachings of this disclosure. Suitable DNA components, selectablemarker genes, reporter genes, enhancers, introns, and the like aredescribed by K. Weising et al., Ann. Rev. Genetics, 22, 421 (1988).

Typically, the structural nucleic acid sequence of interest and 5′and/or 3′ tobacco osmotin UTRs are inserted into an appropriate cloningvector at appropriate restriction site(s) such that the structural geneof interest is operably linked to a desired promoter and the 5′ and/or3′ tobacco osmotin UTRs are functionally linked to the structuralnucleic acid sequence of interest. In preparing the gene constructs ofthis invention, the various nucleic acid fragments may be manipulated,so as to provide for the nucleic acid sequences in the properorientation and, as appropriate, in the proper reading frame. Of course,adapters or linkers may be employed for joining nucleic acid fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like.

The expression of structural genes employed in the present invention maybe driven by any number of promoters. Although the endogenous promoterof a structural gene of interest may be utilized herein fortranscriptional regulation of the gene, preferably, the promoter is aforeign regulatory sequence. For plant expression vectors, suitableviral promoters include the Cassaya Vein Mosaic Virus promoter(Verdaguer et al., Plant Mol. Biol. 31(6):1129-39 (1996); 35S RNA and19S RNA promoters of Cauliflower Mosaic Virus (CaMV) (Brisson et al.,Nature 310:511 (1984); Odell et al., Nature, 313:810 (1985); theenhanced and double enhanced CaMV35S promoter (Kay et al., Science236:1299-1302 (1987); the full-length transcript promoter from FigwortMosaic Virus (FMV) (Gowda et al., J. Cell Biochem., 13D: 301, 1989) andthe coat protein promoter from TMV (Takamatsu et al., EMBO J. 6:307,1987). Other useful promoters include the light-inducible promoter fromthe small subunit ribulose 1,5-bisphosphate carboxylase oxygenase(ssRUBISCO) (Coruzzi et al., EMBO J., 3:1671 (1984); Broglie, et al.,Science 224:838 (1984); rice actin promoter (McElroy et al., Plant Cell.2(2):163-71 (1990); and Adh1 promoter (Dennis et al., Nucleic Acids Res.12(9):3983-4000 (1984)); mannopine synthase promoter (Velten et al.,EMBO J., 3:2723, 1984); nopaline synthase (NOS) and octopine synthase(OCS) promoters (carried on tumor-inducing plasmids of Agrobacteriumtumefaciens) or heat shock promoters, e.g., soybean hsp17.5-E orhsp17.3-B (Gurley et al., Mol. Cell. Biol. 6:559 (1986); Severin et al.,Plant Mol. Biol. 15:827, (1990)).

Analysis of the cloning steps are typically conducted and may involvesequence analysis, restriction analysis, electrophoresis, or the like.After each manipulation the DNA sequence to be used in the finalconstruct may be restricted and joined to the next sequence, where eachof the partial constructs may be cloned in the same or differentplasmids.

Once the cloning steps have been completed, various techniques existwhich allow for the introduction, plant regeneration, stableintegration, and expression of foreign recombinant vectors containingheterologous genes of interest in plant cells. One such techniqueinvolves acceleration of microparticles coated with genetic materialdirectly into plant cells (U.S. Pat. No. 4,945,050 to Cornell; U.S. Pat.No. 5,141,131 to DowElanco; and U.S. Pat. Nos. 5,538,877 and 5,538,880,both to Dekalb). This technique is commonly referred to as“microparticle bombardment” or “biolistics”. Plants may also betransformed using Agrobacterium technology (U.S. Pat. No. 5,177,010 toUniversity of Toledo, U.S. Pat. No. 5,104,310 to Texas A&M, EuropeanPatent Application 0131624B1, European Patent Applications 120516,159418B1 and 176,112 to Schilperoot, U.S. Pat. Nos. 5,149,645,5,469,976, 5,464,763 and 4,940,838 and 4,693,976 to Schilperoot,European Patent Applications 116718, 290799, 320500 all to Max Planck,European Patent Applications 604662, 627752 and U.S. Pat. No. 5,591,616to Japan Tobacco, European Patent Applications 0267159, and 0292435 andU.S. Pat. No. 5,231,019 all to Ciba-Geigy, U.S. Pat. Nos. 5,463,174 and4,762,785 both to Calgene, and U.S. Pat. Nos. 5,004,863 and 5,159,135both to Agracetus). Another transformation method involves the use ofelongated needle-like microfibers or “whiskers” to transform maize cellsuspension cultures (U.S. Pat. Nos. 5,302,523 and 5,464,765 both toZeneca). In addition, electroporation technology has been used totransform plant cells from which fertile plants have been obtained (WO87/06614 to Boyce Thompson Institute; U.S. Pat. Nos. 5,472,869 and5,384,253 both to Dekalb; U.S. Pat. Nos. 5,679,558, 5,641,664, WO9209696and WO9321335 to Plant Genetic Systems).

Still further techniques for the transformation of plant cells include:direct DNA uptake mechanisms (see Mandel and Higa, J. Mol. Biol.,53:159-162 (1972); Dityatkin et al., Biochimica et Biophysica Acta,281:319-323 (1972); Wigler et al., Cell, 16:77 (1979); and Uchimiya etal., In: Proc. 5th Intl. Cong. Plant Tissue and Cell Culture, A.Fujiwara (ed.), Jap. Assoc. for Plant Tissue Culture, Tokyo, pp. 507-508(1982)); fusion mechanisms (see Uchidaz et al., In: Introduction ofMacromolecules Into Viable Mammalian Cells, Baserga et al. (eds.) WistarSymposium Series, 1:169-185 (1980)); site specific recombination (seeWO/9109957), and various infectious agents (see Fraley et al., CRC CritRev. Plant Sci., 4: 1-46 (1986); and Anderson, Science, 226:401-409(1984)).

The appropriate procedure to transform a selected plant cell may bechosen in accordance with the plant cell used. Based on the experienceto date, there appears to be little difference in the expression ofgenes, once inserted into cells, attributable to the method oftransformation itself. Rather, the activity of the foreign gene insertedinto plant cells is dependent upon the influence of endogenous plant DNAadjacent the insert. Generally, the insertion of heterologous genesappears to be random using any transformation technique; however,technology currently exists for producing plants with site specificrecombination of DNA into plants cells (see WO91/09957.

The particular methods used to transform such plant cells are notcritical to this invention, nor are subsequent steps, such asregeneration of such plant cells, as necessary. Any method orcombination of methods resulting in the expression of the desiredsequence or sequences under the regulatory control of one or more of thesubject 5′ and/or 3′ UTRs is acceptable.

Once introduced into the plant tissue, the expression of the structuralgene may be assayed in a transient expression system, or it may bedetermined after selection for stable integration within the plantgenome.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler et al., Cell 11:223 (1977)) and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes thatcan be employed in tk⁻ or aprt⁻ cells, respectively. Also,antimetabolite, antibiotic, or herbicide resistance can be used as thebasis for selection; for example, dhfr, which confers resistance tomethotrexate (Wigler et al., Proc. Natl. Acad. Sci., 77:3567 (1980));npt, which confers resistance to the aminoglycosides neomycin and G-418(Colbere-Garapin et al., J. Mol. Biol., 150:1)(1981)); and ALS (U.S.Pat. No. 5,378,824 to Bedbrook) or PAT (Wehrmann et al., Nat Biotechnol14(10):1274-8 (1996)), which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively. Additional selectablegenes have been described, for example, trpB, which allows cells toutilize indole in place of tryptophan, or hisD, which allows cells toutilize histinol in place of histidine (Hartman and Mulligan, Proc.Natl. Acad. Sci., 85:8047 (1988)). More recently, the use of visiblemarkers has gained popularity with such markers as GFP, anthocyanins,α-glucuronidase and its substrate GUS, luciferase and its substrateluciferin, being widely used not only to identify transformants, butalso to quantify the amount of transient or stable protein expressionattributable to a specific vector system (Rhodes et al., Methods Mol.Biol., 55:121 (1995)).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression mayneed to be confirmed. For example, if the sequence encoding apolypeptide is inserted within a marker gene sequence, recombinant cellscontaining sequences encoding the polypeptide can be identified by theabsence of marker gene function. Alternatively, a marker gene can beplaced in tandem with a sequence encoding the polypeptide under thecontrol of a single promoter. Expression of the marker gene in responseto induction or selection usually indicates expression of the tandemgene as well.

Alternatively, host cells that contain the nucleic acid sequenceencoding the polypeptide of interest (for example, a polypeptide encodedby a nucleic acid of the present invention) and express the polypeptidemay be identified by a variety of procedures known to those of skill inthe art. These procedures include, but are not limited to, DNA-DNA orDNA-RNA hybridizations and protein bioassay or immunoassay techniquesthat include membrane, solution, or chip based technologies for thedetection and/or quantification of nucleic acid or protein.

The presence of polynucleotide sequences encoding a polypeptide ofinterest (for example, a polypeptide encoded by a nucleic acid of thepresent invention) can be detected by DNA-DNA or DNA-RNA hybridizationor amplification using probes or portions or fragments ofpolynucleotides encoding the polypeptide. Nucleic acid amplificationbased assays involve the use of oligonucleotides or oligomers based onthe sequences encoding the polypeptide to detect transformantscontaining DNA or RNA encoding the polypeptide. As used herein“oligonucleotides” or “oligomers” refer to a nucleic acid sequence of atleast about 10 nucleotides and as many as about 60 nucleotides,preferably about 15 to 30 nucleotides, and more preferably about 20-25nucleotides, that can be used as a probe or amplimer.

A variety of protocols for detecting and measuring the expression of apolypeptide (for example, a polypeptide encoded by a nucleic acid of thepresent invention), using either polyclonal or monoclonal antibodiesspecific for the protein are known in the art. Examples includeenzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), andfluorescence activated cell sorting (FACS). A two-site, monoclonal-basedimmunoassay utilizing monoclonal antibodies reactive to twonon-interfering epitopes on the polypeptide is preferred, but acompetitive binding assay may be employed. These and other assays aredescribed, among other places, in Hampton et al., Serological Methods, aLaboratory Manual, APS Press, St Paul, Minn. (1990), and Maddox et al.,J. Exp. Med., 158:1211 (1983).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding a polypeptide ofinterest include oligonucleotide labeling, nick translation,end-labeling or PCR amplification using a labeled nucleotide.Alternatively, the sequences encoding the polypeptide, or any portionsthereof may be cloned into a vector for the production of an mRNA probe.Such vectors are known in the art, are commercially available, and maybe used to synthesize RNA probes in vitro by addition of an appropriateRNA polymerase such as T7, T3, or SP6 and labeled nucleotides. Theseprocedures may be conducted using a variety of commercially availablekits from Pharmacia & Upjohn (Kalamazoo, Mich.), Promega Corporation(Madison, Wis.) and U.S. Biochemical Corp. (Cleveland, Ohio). Suitablereporter molecules or labels, that may be used, include radionuclides,enzymes, fluorescent, chemiluminescent, or chromogenic agents as well assubstrates, cofactors, inhibitors, magnetic particles, and the like.

Techniques are known for the in vitro culture of plant tissue, and, in anumber of cases, for regeneration into whole plants. The appropriateprocedure to produce mature transgenic plants may be chosen inaccordance with the plant species used. Regeneration varies from speciesto species of plants. Efficient regeneration will depend upon themedium, on the genotype, and on the history of the culture. Once wholeplants have been obtained, they can be sexually or clonally reproducedin such a manner that at least one copy of the sequence is present inthe cells of the progeny. Seed from the regenerated plants can becollected for future use, and plants grown from this seed. Proceduresfor transferring the introduced gene from the originally transformedplant into commercially useful cultivars are known to those skilled inthe art.

Particular embodiments of this invention are further exemplified in theExamples. However, those skilled in the art will readily appreciate thatthe specific experiments detailed are only illustrative of the inventionas described more fully in the claims which follow thereafter.

The preferred gene of interest for use in the present invention is theToxin A gene from Photorabadus luminescens (hereinafter “Photorabadus”or “p. luminescens”).

EXAMPLES Experimental Design

Photorhabdus luminescens is a gram-negative bacterium that formsentomopathogenic symbioses with Heterorhabditis spp. soil nematodes.(ffrench-Constant et al., Cell Mol Life Sci 57(5):828-33 (2000);ffrench-Constant et al. Curr Opin Microbiol. 2(3):284-8 (1999)).Nematodes harboring this bacterium have long been used as biologicalcontrol agents for insect infestation. After the nematode invades theinsect host, the bacteria are released into the insect haemocoel wherethey produce toxins and proteases that kill the insect host and renderthe host cadaver into a ready source of nutrients for both bacteria andnematode growth.

Several groups of toxin complexes have been purified from P. luminescensand their corresponding genes have been cloned. (Bowen et al., Science280:2129-32 (1998); Merlo et al., GenBank Accession No. AF188483(1999)). In previously conducted work, it was found that thefermentation broth of P. luminescens strain W-14 contains at least twopotent proteins, Toxin A and Toxin B, which independently contribute tothe insecticidal activity against Southern corn rootworm (SCR;Diabrotica undecimpunctata howardi) and tobacco hornworm (THW; Manducasexta). (Gou et al., J. Biol. Chem. 274(14):9836-42 (1999)). Theactivities of these two proteins differ dramatically in toploadedartificial diet assays. LD₅₀ values (lethal dose for 50% of insects)against SCR are 5 ng/cm² diet and 87 ng/cm² diet for Toxin A and ToxinB, respectively.

The 283 kD Toxin A protein (SEQ. ED. No. 4) (designated A0 proteinherein) of P. luminescens strain W-14 is encoded by a single openreading frame (designated tcdA) of 7548 bp. (GenBank Accession No.AF188483; Gou, 1999). In the bacterial fermentation broth, native ToxinA exists in a large complex (>860 kD) consistent in size with ahomotetramer (Verdaguer et al., Plant Mol. Biol. 31 (6):1129-39 (1996)).Isolation and characterization (N-terminal sequencing and MALDI-TOF/QTOFanalyses) of the proteins comprising the Toxin A complex revealed thatthe N-terminal 88 amino acids of the A0 primary gene product areremoved, and the remaining peptide is cleaved into two largepolypeptides, designated A1 (5.8 kb) and A2 (1.7 kb) herein. During thisprocessing step, another 88 internal amino acids are lost. (See FIG. 1).The order of these cleavage steps, and the significance of theN-terminal and internal deletions relative to toxin activity arebelieved to be unknown in the art at the time of this disclosures. Ithas also previously been unclear as to whether the A1 polypeptide aloneis responsible for the insecticidal activity of Toxin A.

To assess the potential use of various forms of the Toxin A gene forpest control, its insecticidal activity was tested in transgenicArabidopsis plants as follows.

Six plant transformation vectors (pDAB7031-pDAB7036) were constructedwhich contained various forms of the Toxin A gene under the control of aconstitutive Cassaya Vein Mosaic Virus promoter (CsVMV). (See FIG. 2).These Toxin A gene fragments included: 1) full-length A0 gene (A0, 7.5kb) (SEQ. ID. No. 5) in construct pDAB7031, 2) A0 gene with N-terminaltruncation (A0/ΔN, 7.3 kb) (SEQ. ID. No. 6) in construct pDAB7032, 3)full-length A1 gene (A1, 5.8 kb) (SEQ. ID. No. 7) in construct pDAB7033,4) A1 gene with C-terminal truncation (A1/ΔC, 5.6 kb) (SEQ. ID. No. 8)in construct pDAB7034, 5) A1 gene with both N- and C-terminaltruncations (A1/ΔN+ΔC, 5.4) (SEQ. ID. No. 9) in construct pDAB7035, and6) full-length A2 gene (A2, 1.7 kb) (SEQ. ID. No. 10) in constructpDAB7036. These six constructs were transformed into Arabidopsis plantsvia Agrobacterium-mediated transformation. Transgenic plants wereselected based on the phenotype of kanamycin resistance.

As a strategy to enhance the expression of Toxin A in plant cells,additional gene constructs were also engineered such that three of ToxinA gene fragments A0 (SEQ. ID. No. 5), A1/ΔC (SEQ. ID. No. 8), and A2(SEQ. ID. No. 10) were flanked on respective ends by 5′ and 3′ UTRsequences (SEQ. ID. Nos. 2 and 3, respectively) isolated from a tobaccoosmotin gene. The resulting constructs were designated pDAB7026, inpDAB7027, pDAB7028, respectively (see FIG. 7). The protein expressionlevels of pDAB7026, pDAB7027 and pDAB7028 (hereinafter, the “osmotinUTR-Toxin A constructs”) were then compared with Toxin A constructs notcontaining the osmotin UTRS—pDAB7031 through pDAB7036 (see FIG. 2)(hereinafter, the “non-osmotin UTR-Toxin A constructs”).

Plasmid Construction

Unless otherwise noted herein, standard methods of DNA purification,restriction enzyme digestion, agarose gel analysis, DNA fragmentisolation, ligation and transformation may be used as described inSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press (2d ed., 1989)) and Ausubel et al., CurrentProtocols in Molecular Biology (New York: John Wiley and Sons) (1987).

The 7548 bp DNA sequence of the native Toxin A gene ORF used in thiswork was determined from a gene cloned in this laboratory from P.luminescens strain W-14. (GenBank Accession No. AF188483). An alaninecodon was added at the second position of the ORF to accommodate an NcoI recognition site spanning the start codon. The resulting nucleic acidsequence, which coded for essentially the same protein, was subsequentlydesigned according to parameters outlined in Adang et al., Plant Mol.Biol. 21 (1993). Synthesis of gene fragments and assembly into an intactcoding region were performed by Operon Technologies (Alameda, Calif.).Rebuilding of the Toxin A gene removed putative RNA instabilitysequences (ATTTAA), potential intron splice signals, and potentialpolyadenylation signal sequences, and adjusted codon usage toaccommodate expression in both monocot and dicot plant species (“plantoptimized”) (see PCT Application WO 01/11029, hereby incorporated byreference herein in its entirety). Unique Nco I and Sac I sites wereadded to the 5′ and 3′ ends of the coding region, respectfully. Variousgene derivatives with corresponding Nco I and Sac I sites were generatedfrom the basic tcdA coding region using PCR methods: A0/ΔN gene (SEQ ID.No. 6), A1 gene (SEQ ID. No. 7), A1/ΔC gene (SEQ ID. No. 8), A1/ΔN+ΔCgene (SEQ ID. No. 9) and A2 gene (SEQ ID. No. 10). All Toxin A genefragments were thus placed under the expression control of the CsVMVpromoter and a 3′UTR/polyadenylation signal sequence derived from theintergenic region between ORFs25/26 of Ti plasmid pTi-15955 (Barker etal., Plant Mol. Biol. 2, 335 (1983)). Each Toxin A gene expressioncassette was excised (Asc I and Pme I) and cloned between the T-DNAborders on binary vector PDAB1542 (FIG. 12), which contains a kanamycinresistance gene as selectable marker for plant transformation. Theresultant constructs (pDAB7031 through pDAB7036) are further describedherein and diagrammed in FIG. 2.

A control plant transformation vector (pDAB7029) containing aCsVMV-GUS-ORF25 expression cassette (Jefferson, Plant Molec. Biol. Rep.5, 387 (1987)) was constructed using the same strategy. The GUS gene wasfirst excised from plasmid pKA882 (FIG. 13) by Nco I and Sac I enzymesand inserted in the place of the PAT gene in plasmid pDAB7013 (FIG. 14).The CsVMV-GUS-ORF25 cassette was then moved into binary vector pDAB1542(FIG. 12) using enzymes Asc I and Pme 1.

To generate osmotin-Toxin A gene constructs, a pair of complementaryoligonucleotides encoding the 40 bp 5′ UTR sequence of a tobacco osmotingene (SEQ. ID No. 1) was chemically synthesized according to thepublished sequence (Nelson et al., 1992) except for a modification whichchanged a single “atg” codon to “att” so as to remove a putativeinitiation codon from the 5′ UTR sequence (resulting in SEQ. ID No. 2).During the synthesis, Bgl II and Nco I sites were added to 5′ and 3′ends. The resulting 5′ UTR sequence was then inserted into the same sitebetween CsVMV promoter and PAT gene in vector pDAB7013 (FIG. 14),resulting in plasmid pDAB7020 (FIG. 15). The 3′ UTR sequences of thesame tobacco osmotin gene (SEQ. ID No. 3) were PCR amplified from anosmotin cDNA clone (Liu et al., Proc. Natl. Acad. Sci. U.S.A.91:1888-1892 (1994); a kind gift from Dr. Ray Bressan, PurdueUniversity). During the PCR amplification, Sac I and Xho I sites wereadded to the 5′ and 3′ ends, respectively. The amplified osmotin 3′ UTRsequence (SEQ. ID. No. 3) was then used to replace the ORF25/26 3′sequence on pDAB6001 (FIG. 16, giving rise to plasmid pDAB7002 (FIG.17). The PAT gene and osmotin 3′ UTR were the excised from pDAB7002 withNco I and Xho I and used to replaced the GUS gene on pDAB7020, resultingplasmid pDAB7021 (FIG. 18), which contains CsVMV-OSM (osmotin) 5′UTR/PAT/OSM 3′ UTR-ORF25/26 expression cassette. The coding regions ofthree Toxin A gene fragments, A0 (SEQ. ID. No. 5), A1/ΔC (SEQ. ID. No.5), A1/ΔC (SEQ. ID. No. 8), and A2 (SEQ. ID. No. 10), were then used toreplace the PAT gene on pDAB7021 (FIG. 18). Finally, the expressioncassettes containing the modified coding region fragments under controlof the CxVMV promoter and ORF25/26 3′ UTR were cloned separately intobinary vector pDAB1542 (FIG. 12).

A map of a plant transformation vector containing the full-length A0gene with ostmotin 5′ and 3′ UTR (pDAB7026) is shown in FIG. 19. Using asimilar strategy, a control plant transformation vector containingCsVMV-OSM 5′UTR/GUS/OSM 3′UTR-ORF 25/26 was also constructed.

In addition to the vector embodiments described above, one of skill inthe art will recognize that a generic vector can readily be createdwhich will allow any gene of interest to be cloned adjacent the 5′ and3′ tobacco osmotin UTSs of the present invention. As a non-limitingexample, the plant expression vector pBI121 (Clontech Laboratories, PaloAlto, Calif.) contains an expression cassette of the GUS reporter genedriven by a CaMV 35S promoter and terminated by NOS terminator.(Jefferson, Nature 342:837-838, 1989). There are three restrictionsites, Xba I, BamH I, and Sma I, between the CaMV 35S promoter and GUSreporter gene. Vector pBI121 also has a Sst I site between the GUS geneand its NOS terminator sequence. A 5′ osmotin UTR sequence of thepresent invention may be chemically synthesized with Xba I and BamH Isites on its 5′ and 3′ ends using standard techniques, and then readilyinserted into the Xba I and BamH I sites on pBI121. To insert theosmotin 3′ UTR sequence into vector pBI121, PCR amplification procedurescan be used to isolate the osmotin 3′ UTR from osmotin cDNA cone withthe addition of Sma I and Sst I sites to its 5′ and 3′ ends. Thisosmotin 3′UTR sequence then can be inserted into the Sma I and Sst Isites on pBI121, which will replace the GUS coding region. This cloningstep creates an expression cassette of CaMV 35S-OSM 5′UTR/OSM 3′UTR-NOSon the resultant plasmid. In this expression cassette, there will be tworestriction sites, BamHI and SmaI, between osmotin 5′ UTR and 3′ UTRsequence. Thus, using BamH I and Sma I restriction enzymes and theappropriate restriction sites on the gene of interest, an gene ofinterest may be cloned into pBI121 for expression in transgenic plants.

Plant Growth and Transformation

Arabidopsis plants (Columbia ecotype) were grown at 22° C. with alighting cycle of 16 hours light and 8 hours dark. All planttransformation constructs were transformed into Agrobacterium strain C58(Z707) (ATCC 33970) using either electroporation (Mattanovich et al.,Efficient transformation of Agrobacterium spp. by electroporation.Nucleic Acids Research 17(16) pp 6747 (1989); Mersereau et al.,Efficient transformation of Agrobacterium tumefaciens byelectroporation. Gene (90) pps 149-151 (1990)) or freeze-thaw methods(Hofgen and Willmitzer, Storage of competent cells for Agrobacteriumtransformation. Nucleic Acids Research, 16:9877 (1998). Planttransformations were performed using the vacuum infiltration method(Bechtold et al., Mol. Biol. Genet. 316: 1194-1199 (1993)). Transgenicplants were selected based on the phenotype of kanamycin resistance.

Northern Hybridization

Total RNA was extracted from 150 mg mature leaf tissues using RNeasyMini Plant Kit (QIAGEN, Inc, Valencia, Calif.). For RNA blot analysis, 5ug of total RNA was loaded onto 1.5% agarose gels containingformaldehyde and processed for Northern analysis. Hybridizations wereperformed at 42° for 4 hours in a UL TRAhyb solution (Ambion, Inc.,Austin, Tex.). After hybridization, membranes were washed twice with2×SSPE, 0.5% SDS for 15 min, then twice with 0.1×SSPE, 0.1% SDS. Thefirst three washes were at room temperature, and the final wash was at42° C.

Bioassay of Transgenic Plants Against THW

Tobacco hornworm (THW, Manduca sexta) eggs were received from the NorthCarolina State University insectary. Eggs were incubated in lightedchambers (Percival, Boone, Iowa) at 22° or 28° C. for 2 to 3 days in 90mm Petri dishes with agar solution. The morning of the bioassay, anylarvae that hatched overnight were removed from the plates, and onlyfresh larvae were used in the bioassay (less than 6 hours old,preferably). 128-well CD International (Pitman, N.J.) bioassay trayswere prepared by placing 0.5 ml of a 2% agar solution into each well.Arabidopsis leaves were taken from 5-week-old plants. For each plant,leaf tissues were distributed evenly among 8 wells. A single neonatehornworm larva was placed into each well. Wells were covered withperforated sticky lids and the insects were allowed to feed for threedays (72 hr) in a chamber at 28° C. and 16:8 light:dark cycle. After 72hours, insect mortality and weight scores were recorded. Mortality indexwas determined from the number of dead larvae out of the total numberfor any given plant. Two control (GUS) plants were used for every 16transgenic plants with the Toxin A gene construct. Data analysis wasperformed on insect mortality scores by comparing the percent insectmortality on experimental versus control plants. Mortality scores weretransformed and a z-test was used. Plants that showed “moderate to high”Toxin A protein levels and significantly higher mortality than thecontrols (at p=0.05) were considered “active.” If the Toxin A proteinlevel was low, but mortality was high (usually >50%), plants werere-tested (usually in the next generation, but the original plant may bere-sampled). To reveal any potential growth inhibition effects,individual insect weights were analyzed by ANOVA, comparing transformedplants with controls.

Experimental Results

Non-Osmotin UTR-Toxin A Constructs

Northern hybridization experiments indicated that 67% of the T₀ plantsexamined (43 of 64) showed a single Toxin A RNA species of the expectedsize (FIG. 2). So far as we are aware, the full-length tcdA transcriptis the largest transgenic RNA produced in plants to date. RNA expressionlevels varied from line to line (data not shown). Immunoblot (western)analysis was performed to examine the protein expression patterns inplants for each construct. Purified Toxin A protein produced from arecombinant E. coil strain, which exhibited three bands (A0, A1/ΔC, andA2 proteins) on SDS-PAGE gel, was used as a positive standard. Plantstransformed with the CsVMV-GUS-ORF25 construct (pDAB7029) served as thenegative control. The results (FIGS. 3A and 3B) showed that plantscarrying construct pDAB7031 (full-length tcdA) (FIG. 20) produced threeprotein bands which aligned with the positive control (FIG. 3A). InpDAB7036 plants, only a single protein band was observed, whosemolecular weight was slightly smaller than the full-length A0 proteindue to the N-terminal truncation of 88 amino acids (FIG. 3B). Theabsence of detectable A1/ΔC, and A2 proteins suggests that the 88 aminoacids at the N-terminus of the A0 protein act as a control signal forinternal processing between the two large subunits. From plants carryingthe three A1 gene constructs, single A1 protein bands with the expectedmolecular sizes were found (FIG. 3C). The A1 protein in pDAB7035 plantswas the same size as that of the standard (FIG. 3A), while A1 proteinsfrom pDAB7033 and pDAB7034 plants had slightly smaller sizes due to thetruncations at the C-terminus or both ends (FIGS. 5B and 5C). Incontrast, no A2 protein was detected in 32 examined plants transformedwith construct pDAB7032 (data not shown), even though A2 RNA wasproperly produced (FIG. 2). These results suggest that either thetranslation efficiency of A2 RNA is extremely low, the A2 protein isvery unstable in plants, or the A2 protein is not extracted by themethodology used.

Protein accumulation levels were further quantified for each primarytransformant using an ELISA procedure (FIG. 28—Table 1). For constructpDAB7031 (FIG. 20), all the positive lines except lines 7031-43 and7031-25 contained Toxin A protein at levels lower than 200 ppm (partsper million; in this work, 1,000 ppm is equal to 0.1% of totalextractable protein).

The T₁ selfed progeny from three pDAB7031 lines (7031-043, 025, and 041)were tested for Toxin A protein accumulation and for resistance to THWfeeding. All progeny analyzed in this work were pre-selected onkanamycin-containing medium to ensure the presence of transgenes.Parents of these three lines had shown Toxin A protein levels of 1056,349, and 134 ppm, respectively, at the T₀ generation. However, none ofthe progeny from lines 7031-025 and 7031-041 produced Toxin A protein.For line 7031-043, 9 out of 32 progeny showed very low levels of Toxin Aprotein (<80 ppm); none of the others showed detectable Toxin A.Apparently, Toxin A gene expression was silenced in these progenyplants. As a consequence, none of these plants showed resistance to THW(data not shown).

These data prompted a larger scale examination of primary transformants(T₀ plants) for insecticidal activity and Toxin A accretion levels. Forthis purpose, another 280 transgenic lines were generated with constructpDAB7031 (FIG. 20). Among these 280 transformants, only one line(7031-240) showed significantly high insecticidal activity (100% insectmortality) (FIG. 29—Table 2). This plant had a Toxin A protein level of788 ppm, and was the highest expresser among these 280 lines. Toxin Aprotein production and insect activity of 32 of its T₁ progeny weredetermined. The results showed that 11 progeny had undetectable levelsof Toxin A protein and insecticidal activity, while the other 21 plantsretained the high to very high levels of Toxin A protein production(739-7023 ppm) and showed 100% insect mortality. Insect mortality forthe control plants transformed with construct CsVMV-GUS-ORF25 was 18.7%.

The above results indicated that when Toxin A accumulation reached athreshold level in plants (about 700 ppm), it could confer completeresistance to THW. However, it was noticed that the frequency ofrecovering insect-resistant lines was very low (1 out of 340 or 0.3%).This was probably due to the overall low expression of the large Toxin Agene from transformation vector pDAB7031 (FIG. 20), even though a strongconstitutive promoter was used.

Osmotin UTR-Toxin A Constructs

To enhance expression of the Toxin A gene in plants, new constructs wereproduced by adding 5′ and 3′ UTR sequences (SEQ. ID. Nos. 2 and 3) froma tobacco osmotin gene to the corresponding ends of the Toxin A codingregions as described herein. To determine if these osmotin UTRstructural elements would improve Toxin A gene expression levels, theireffects were tested in three Toxin A gene constructs: pDAB7026(full-length A0 gene) (SEQ. ID. No. 5), pDAB7027 (A1/ΔC gene) (SEQ. ID.No. 8), and pDAB7028 (A2 gene) (SEQ. ID. No. 10). (See FIG. 7).

RNA expression patterns were examined in transgenic plants transformedwith these three osmotin-Toxin A gene constructs. Northern blot analysesindicated 20 out of 30 examined plants, which covered all threeconstructs, showed a single species of Toxin A RNA with the expectedmolecular size (data not shown). These results were therefore the sameas those observed from analogous non-osmotin UTR-Toxin A constructs.Toxin A protein levels of these osmotin-Toxin A constructs were comparedwith their nonosmotin-Toxin A counterparts (FIG. 29—Table 2). Of 340total pDAB7031 plants, only 23% had detectable levels of Toxin Aprotein, and the average Toxin A accumulation level of expressing plantswas 67 ppm. For the 273 pDAB7026 transgenic plants, 39% contained ToxinA protein, and the average level of the expressing plants was 390 ppm.Therefore, there was about a 6-fold difference between these twoconstructs. If all plants examined were included in the statisticalanalysis (i.e. expressers and non-expressers), the average Toxin Aproduction levels were 15 ppm for the pDAB7031 plants, and 150 ppm forthe pDAB7026 plants (10-fold difference). In addition to an increase inoverall Toxin A producers and Toxin A accumulation, there was a alsodifference in the number of high expressers (Toxin A protein >700 ppm)for each construct. Among the pDAB7031 plants, there were 2 highexpressers (0.6%), whereas in the pDAB7026 plant group, there were 13high expressers (4.7%). For some high expressers with construct pDAB7026(>2,000 ppm), the accumulation of Toxin A protein in plant cells couldbe easily observed in an SDS-PAGE gel (FIG. 8B).

For plants carrying construct pDAB7033, 58% produced the truncated A1proteins, and the average A1 protein level of expressing plants was 251ppm. For pDAB7028 transgenic plants, 90% of examined plants showed A1protein, and the average level of accumulation was 1131 ppm (4.5-foldincrease). The effect of the osmotin flanking sequences was alsoobserved in transgenic plants carrying constructs designed to producethe A2 protein. There was no detectable A2 protein in any of the 32pDAB7032 plants examined (Table 1). However, in the 25 plants carryingconstruct pDAB7028, 40% (10 plants) produced a single band of A2 protein(FIG. 29—Table 2 and FIG. 8A), although the overall expression level wasnot high. These data clearly demonstrate that tobacco osmotin UTRsequences can greatly enhance Toxin A gene expression in transgenicArabidopsis plants.

The increase in the overall accumulation of Toxin A protein alsoincreased the chances of recovering insect-resistant lines. Bioassayswere performed directly on 259 primary pDAB7026 transformants. Amongthese T₀ plants, 9 lines (not including line 7026-011, see below) showed100% insect mortality (FIG. 30—Table 3). Except for line 7026-127, allthese lines had Toxin A levels higher than 1,000 ppm. The bioactivityand high level accumulation of the Toxin A protein were coordinatelytransmitted into the next generation. At least 32 progeny were examinedfor each of these 9 lines as well as line 7026-011 (FIG. 30—Table 3).Although bioassays were not done on the T₀ plant of line 7026-011, itsT₁ progeny were included in this study because the T0 plant had a highlevel of Toxin A protein. For line 7026-011, all except one of the 71 T₁progeny showed 100% insect mortality. The remaining plant showed 87.5%mortality (1 of 8 insects survived), which was neverthelesssignificantly higher than the control mortality (19%) (Table 6). Incontrast, for line 7026-195, none of its 32 T₁ progeny showed Toxin Aaccumulation or insecticidal activity. For the other lines, thepercentage of progeny that showed high levels of Toxin A protein andinsecticidal activity ranged from 90% to 18% (FIG. 30—Table 3). Intotal, 333 T₁ progeny for these 10 lines were analyzed, and 214 werefound to retain a high level of Toxin A protein. Among these T₁ highexpressers, 211 (98%) had significantly high insecticidal activity whencompared to the control group (FIG. 30—Table 3). FIG. 31—Table 4 showsthe average insect mortality of the high expressers and the low ornon-expressers among the T₁ progeny for each line. These results furtherconfirmed that the high level of accumulation of Toxin A protein wasresponsible for the plants' insecticidal activity against THW.

During the screening for insect-resistant lines amongst T₀ pDAB7026plants, four lines were found that had very low or no Toxin Aaccumulation, yet showed a significantly high insecticidal activity.Sixteen T₁ progeny from each of these four lines were examined. None ofthese plants showed any Toxin A protein or insect activity (data notshown), suggesting that these four lines were false positives in theinsect bioassays. The earlier bioassay results on the T₀ plants wereprobably due to variations in test insect viability or to someundetermined artifacts resulting from the transformation processes.

In contrast, we also identified four lines that showed high levelaccumulation of Toxin A protein, but no significant insect mortality. Todetermine if these could be false negative results, 32 progeny from eachof these lines were analyzed. Except for line 7026-101, in which none ofthe progeny showed any Toxin A protein or insect activity, the otherthree lines, with a total 11 progeny, showed high level expression ofthe Toxin A gene (FIG. 32—Table 5). The average insect mortality shownby these T₁ high expressers was 98.2% while the control group was 14.0%.These results confirmed that the non-significant activity observed onthese lines at the T₀ generation could have been escapes of the currentbioassay procedure. These aberrant results underscore the need toexamine transgenic progeny, rather than solely T0 plants, in assessinggene function.

In summary, from 274 pDAB7026 transgenic lines analyzed at the T₁generation, 12 lines (4.4%) were identified with heritable high levelsof Toxin A production and insect activity (FIG. 29—Table 2), even thoughthe degree of heritability varied from 3% (line 7026-101) to 100% (line7026-011). These results demonstrate that the enhanced accumulation ofthe Toxin A protein by the osmotin UTR sequences increased the recoveryfrequency of insect-resistant lines from 0.3% to 4.4% (FIG. 29—Table 2).

Lines 7026-011 and 7026-057 were further followed to their fifthgenerations (T₄ plants) to determine the stability of Toxin A geneexpression and associated insecticidal activity. For line 7026-011, theoverexpression of Toxin A and insecticidal activity were stablymaintained in all progeny for five generations (FIG. 33—Table 6).However, the heritability pattern for line 7026-057 was more complicated(FIG. 9). Thirty-eight T₁ progeny were examined, and all 15 T₁ plantshomozygous for the transgenes had lost Toxin protein production as wellas insect activity. Only 7 of the remaining 23 hemizygous progenyretained both high level accumulation of Toxin A and 100% insectmortality (FIG. 29—Table 2). A T₂ generation was derived from 6 of the 7insect active hemizygous T₁ plants (FIG. 9). The percentage of activeplants for each T₂ family ranged from 0% to 35%.

Examination of T₄ progeny identified one T₃ family in which 60% offamily members were high expressers and were insect active. Oneinteresting question was to determine, for this family, if the averagepercentage of active plants in the T₄ generation would increase over theT₃ generation. Apparently, this was not the case. Similar results werealso seen for the progeny of five T₃ families, which were derived from asingle T₂ family, in which 35.8% family members were active plants.

According to the structural model of the Toxin A complex (FIG. 1), aquestion addressable by these transgenic materials was whether the A2polypeptide is an indispensable part of the complex's activity.Transgenic plants carrying constructs pDAB7033, 7034, and 7035, whichproduce only A1 proteins at relatively high levels, were furtherstudied. From the screening of 146 T₁ progeny, 12 high expressers wereidentified which covered all three A1 constructs. Bioassay resultsshowed that none of these plants showed significantly higher insectmortality compared to the control plants (data not shown). Thisindicated that the A1 protein alone in Arabidopsis is not sufficient forinsecticidal activity against THW.

Discussion

In this work, we first analyzed the expression of a plant-optimized P.luminescens strain W-14 tcdA gene in transgenic Arabidopsis plants. Theresults provided some important insights about the behaviors of Toxin Aprotein in plants: 1) the full length tcdA gene can produce Toxin Aprotein whose final products mimic those observed from the native P.luminescens strain W-14 and from a recombinant E. coli strain,indicating that Toxin A protein is appropriately processed in plantcells; 2) the N-terminal 88 amino acids of the A0 protein seem to serveas a signal peptide for protein cleavage, since the deletion of theseamino acids prevents the cleavage of the TcdA protein into the A1 and A2polypeptides; 3) The N-terminus and C-terminus of the A1 protein werenot further processed in plants cells, otherwise, the A1 proteinsencoded by three different A1 gene constructs would have the samemolecular weights.

In the early stages of this work with construct pDAB7031 (FIG. 20), itwas observed that the overall accumulation of Toxin A protein intransgenic plants was very low. The overall low expression also resultedin a low frequency of recovering insect-resistant lines (0.3% oftransgenics). In the particular case of construct pDAB7032, A2 proteinwas not observed in the transgenic plants, even though the A2 mRNA waseasily detected. Poor transgene expression in plants can be attributedto many factors, especially when using a gene from heterologous sources.Use of a strong promoter does not necessarily guarantee a high level ofgene expression. In addition to low transcriptional activity due tointegration position effects, features such as improper splicing,incomplete polyadenylation, inefficient nuclear export, mRNAinstability, and poor translation efficiency all can result in low levelaccumulation of both mRNA and protein. Elimination of these potentialpitfalls was attempted through complete redesign and synthesis of theplant-optimized Toxin A coding region. Further, Toxin A gene expressionwas enhanced by adding 5′ and 3′ UTR flanking sequences from a tobaccoosmotin gene to the Toxin A gene. Structural features of the osmotinmRNA 5′ and 3′ UTRs are consistent with the criteria of a stable, highlyexpressed plant mRNA: i) the 5′ UTR sequence is highly AT-rich, allowingribosomes to easily scan to the start codon to initiate translation, andii) the 3′ UTR sequences can form a strong stem-loop secondary structurethat may effectively block degradation from RNase. (Kozie, 1996).Indeed, after the osmotin 5′ and 3′ UTR sequences were added to thecorresponding ends of the Toxin A gene(s), the overall production of theA1 and A0 proteins increased 5-10 fold. As a consequence, the recoveryfrequency for insect-resistant lines transformed with tcdA genesincreased from 0.3% to 4.4%. Also, for the first time, accumulation ofthe A2 protein could be detected in 40% of plants examined that weretransformed with the A2 gene alone.

Importantly, it was demonstrated that overexpression of tcdA intransgenic Arabidopsis plants can render the plants completely toxic tofeeding THW. For the first time, it has been clearly shown that thepresence of the A2 subunit is associated with Toxin A's insecticidalactivity, as plants containing only the large A1 subunit were inactive.In our analysis of about 2,500 individual plants, insecticidal activitywas always associated with high level accumulation of the Toxin Aprotein. These results indicate that the Toxin A gene is an excellentcandidate for crop protection in agriculture, since Toxin A also hasstrong activity against SCR. The Toxin A gene and other P. luminescenstoxin genes may open new routes for pest control in agriculture. Untilnow, transgenic crop insect control has heavily relied on the use of Bttoxin genes, and the P. luminescens toxin genes can help reduce theproblem of development of resistance of pests to Bt plants. Stacking theToxin A gene into plants which already contain a Bt gene may alsoincrease the efficacy of insect toxicity in terms of potency and pestspectrum.

SUMMARY

The analysis of protein expression of three Toxin A coding regions intransgenic Arabidopsis plants is provided in Table 1. In plants carryingthe A0 gene construct with no osmotin UTR flanks, only 23% of the 340examined plants showed detectable protein expression. The average ToxinA protein level of expression plants was 67 PPM (parts per million, ngper mg soluble protein). However, for the 273 transgenic plants examinedthat carried the osmotin UTR-A0 gene construct, 39% showed proteinexpression, and the average Toxin A protein level of the expressingplants was 390 PPM. Therefore, osmotin UTR-A0 constructs are expressedabout 6-fold higher as compared to non-osmotin A0 constructs. When allplants examined are included in the statistical analysis, the averageToxin A expression level for the A0 construct was 15 ppm, while theaverage Toxin A expression level for the osmotin UTR-A0 plants was 150PPM. Thus, the difference in average Toxin A production between thesetwo constructs is about 10 fold. The number of high expressers for eachconstruct (Toxin A protein >700 PPM) was also calculated. Among the A0plants, there were 2 high expressers (0.6%), whereas in the osmotinUTR-A0 plant group, 13 high expressers (4.7%) were found.

For transgenic plants carrying the A1/ΔC gene construct, 58% expressedthe truncated A1 proteins, and the average A1 protein expression levelof expressing plants was 251 ppm. For transgenic plants carrying theosmotin UTR-A1/ΔC construct, 90% of examined plants showed A1 proteinexpression, and the average level of expression was 1131 PPM (a 4.5-foldincrease). For transgenic plants carrying the A2 gene, A2 proteinexpression could not be detected in any of the 32 examined plants.However, in 25 osmotin-A2 plants, 10 plants (40%) were found thatexpressed the A2 protein, and the average level for expression was 31pp. These data clearly show that tobacco osmotin UTR sequences cangreatly enhance foreign gene expression in transgenic Arabidopsis plantswith three different gene constructs.

The insecticidal activity of the transgenic plants carrying full-lengthToxin A gene constructs was also evaluated. For non-osmotin/A0 plants,only one line (0.3%) showed complete resistance (100% mortality) againsttobacco hornworm (THW) at the T0 generation, and its activity wasconfirmed at the next generation. For the plant group with the osmotinUTR-A0 gene, 10 lines (3.6%) were found with heritable resistance toTHW.

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the various described embodimentsare merely exemplary of the present invention and that many apparentvariations thereof are possible without departing from the spirit orscope thereof. Accordingly, one skilled in the art will readilyrecognize that the present invention is not limited to the specificembodiments described herein.

1. A method for recombinantly producing a peptide or protein comprising:functionally linking a heterologous promoter and a nucleic acid sequenceconsisting of an isolated osmotin UTR element selected from the groupconsisting of SEQ. ID No. 1 and SEQ. ID No. 2 with a structural gene ofinterest.
 2. A method of increasing expression of a gene in a plant cellcomprising: functionally linking at least heterologous promoter and onenucleic acid sequence consisting of an isolated osmotin UTR elementselected from the group consisting of SEQ. ID No. 1 and SEQ. ID No. 2with a structural gene of interest to create a nucleic acid construct;transforming the plant cell with the nucleic acid construct; and growingthe transformed cell under conditions in which the structural gene ofinterest is expressed.
 3. The method of claim 1, wherein the at leastone structural gene of interest comprises a gene capable of conferring anon-native phenotype in a plant.
 4. The method of claim 1, wherein theat least one structural gene of interest comprises a gene capable ofconferring insecticide or herbicide resistance in a plant.
 5. The methodof claim 1, wherein the at least one structural gene of interestcomprises SEQ. ID. No.
 5. 6. The method of claim 2, wherein the at leastone structural gene of interest comprises a gene capable of conferring anon-native phenotype in a plant.
 7. The method of claim 2, wherein theat least one structural gene of interest comprises a gene capable ofconferring insecticide or herbicide resistance in a plant.
 8. The methodof claim 2, wherein the at least one structural gene of interestcomprises SEQ. ID. No. 5.